Ultrasonic pretreatment assisted enzymolysis for preparation of low molecular weight osteogenic collagen peptides: Kinetics, thermodynamics, and osteogenic activity

Abstract

To prepare low molecular weight (LMW) osteogenic collagen peptides, ultrasonic (US) pretreatment was used to enhance the enzymolysis efficiency of bovine bone collagen (BBC). Therefore, this study analyzed the enzymolysis kinetics, thermodynamics, and osteogenic activity. The kinetic results indicated that US pretreatment increased the association constant (K_a) by 19.30 % and decreased the Michaelis constant (K_m) by 11.14 %. The thermodynamic results indicated that US pretreatment reduced the activation energy (E_a), enthalpy (ΔH), and entropy (ΔS) by 25.73 %, 27.69 %, and 15.39 %, respectively, and Gibbs free energy (ΔG) was reduced by 1.86 (298 K), 1.51 (308 K), 1.18 (318 K), and 0.88 % (328 K), and the reaction rate constant (k) was increased by 89.87 (298 K), 71.54 (308 K), 39.53 (318 K), and 38.44 % (328 K). Structural characterization demonstrated that the shear force, microflow, turbulent force, and cavitation effects produced by US disrupted the compact spatial structure of BBC. Additionally, it resulted in a more uniform distribution of BBC with smaller particles in the solution, that exposed more enzyme cleavage sites. The molecular weight distribution demonstrated that the proportion of collagen peptides less than 1000 Da in UP-BBCP was 83.67 %, which was 14.21 % higher than that (69.46 %) in BBCP. MC3T3-E1 cell experiments demonstrated that UP-BBCP exhibited stronger pro-osteogenic activity than that of BBCP, including proliferation activity (increased by 27.26 %) and calcium deposition level (increased by 35.05 %). In conclusion, US pretreatment may be a potential strategy to enhance enzymolysis efficiency for preparing LMW osteogenic collagen peptides.